Hi Everybody, I am new to immunohistochemistry and immunofluorescence techniques. I need a protocol for freezing brains after paraformaldehye perfusion. I want to cut brains on cryostat. I want to know that how much time brain should be in 4 % PFA solution and 30% sucrose. After that how I can freeze the brains?
Hello Gaurav:
You need to do the following:
1. After perfusion, the brain can be submerged in the same fixative at least for another 24 hrs if not more at 4 C. Cut the tissue in 3 pieces (1 cerebellum, and two pieces from cerebral hemisphere (at least that's what I do for better penetration of the fixative) before submerging them for post fixation step. Make incision before hippocampus so you are not damaging that area (frontal to caudal).
2. Rinse the pieces with chilled 1 X PBS for an hour at 4 C with occasional changes.
3. Add 30% sucrose-PBS solution to them and store at 4 C. The pieces will be floating at the top initially but they will sink to the bottom as they get saturated with sucrose solution.
4. Take them out and drain excess solution. Quick freeze in liquid nitrogen for couple of seconds and make a block with OCT compound either on dry ice or at -80.
5. Gradually change the temp of the block by bringing them to first -40 and then to -20 (so that the block does not break for sudden change in the tissue) before cutting them with the cryostat. Make sure you are turning the instrument on ahead of time and reaching the temp you need to section them. Put the knife inside when you are turning on the machine so it has the same temp as that of the tissue block.
I usually lift the sections and put them in 12 or 24 tissue culture wells and store at 4 C.
Hope it helps. Please reach out if you have any further question on this.
1. After removal of the brain, post-fix in the same fixative for 3-4 days at 4 C.
2. Rinse them in 1 X PBS for an hour with changes in the buffer 3 times at least to remove excess fixative.
3. Put them in 30% Sucrose-PBS solution. The brain will be floating at this point because of the heavyness of the sucrose solution. But eventually it will be totally saturated and sink at the bottom of the tube.
4. Remove them from the sucrose solution and put them in the 2-methylbutane(Isopentane) in a beaker already chilled in liquid nitrogen for 30s-1 m.
5. Remove immediately and put them on dry ice (if the lab is different from your work lab).
6. On the cryotstat tissue holder, drop some OCT compound (already placed on dry ice). In the middle of the freezing (looks whitish), place the tissue in the center of the holder and add some more OCT compound surrounding the tissue. wait for couple of minutes till it becomes solidified. Transfer it inside the cryostat chamber and let the temperature come back to the desired temperature you want to section the tissue.
I usually cut them at -20 to -26 C. Hope it helps.
Good luck. Sorry it is a long procedure but helps preserve the tissue morphology very nicely.
My protocol is very similar to that of Ranu - just shorter
After letting sink in the sucrose, I put the brain on parafilm and remove as much sucrose from the surface as possible using tissue paper
Then I wash in OCT compound to remove the sucrose from the surface and embed the brain in a little plastic mold filled with OCT compound.
This mold I put on dry ice so that the OCT with the brain inside freezes into a block.
This block is then glued to the tissue holder of the cyostat using the same method as Ranu does.
Hope that helps
As an alternative option, to preserve a brain tissue, after perfusion, you can snap freeze your tissue directly in liquid nitrogen, then transfer it to -80 Freezer. This way your proteins will stay intact.
Later on these tissue can be used for cryosectioning
You can post-fix the brains for 2-3 days in the same fixative solution. Then keep the brains in PBS-Sucrose 30% for 48-72 hours, until floating . At this point you can put the brains inside cryostat (cold) and rinse the tissue Tek compound that is a convenient matrix for cryo sectioning. Now put them in parafilm and transfer to freezer.
good luck!
All protocols and suggestions posted describe and resume well the essential steps to follow. Just minor differences regarding to the well described protocol of Ranu.
- May be post fixation does not exceed 48 h to avoid over fixation (not good for antigens).
- After sucrose step finalization, you can keep your brain (or brain sample) in PBS for some days until freezing (in case you can freeze it)
- You can certainly freeze the sample in liquid nitrogen. Freezing it in isopentane (kept at -80°C) could preserve cellular and tissular integrity. At this step, you can stock the brain at -80°C for months until sectioning in cryostat. if you intend to process sectioning, just. transfer the brain fo -20°C, the same to that of cryostat 24 h before.
If I'm freezing tissues in OCT then what is the best container to freeze them in? i.e. should I use the Tissue-Tek cryomolds, or are other containers suitable?
Many thanks for any help here.
I have used similar protocols in order to fix and slice some brains, however the result is still sub-optimal. I keep the slides inside the cryostat chamber while slicing and I thaw each slice after mounting it. When I am done, I place the slides directly in the freezer until further use. Should I let the slides dry overnight before freezing it? I read also that I could use a slide warmer at 37°C for about 30min, has anyone tried that?
Hello Gaurav:
You need to do the following:
1. After perfusion, the brain can be submerged in the same fixative at least for another 24 hrs if not more at 4 C. Cut the tissue in 3 pieces (1 cerebellum, and two pieces from cerebral hemisphere (at least that's what I do for better penetration of the fixative) before submerging them for post fixation step. Make incision before hippocampus so you are not damaging that area (frontal to caudal).
2. Rinse the pieces with chilled 1 X PBS for an hour at 4 C with occasional changes.
3. Add 30% sucrose-PBS solution to them and store at 4 C. The pieces will be floating at the top initially but they will sink to the bottom as they get saturated with sucrose solution.
4. Take them out and drain excess solution. Quick freeze in liquid nitrogen for couple of seconds and make a block with OCT compound either on dry ice or at -80.
5. Gradually change the temp of the block by bringing them to first -40 and then to -20 (so that the block does not break for sudden change in the tissue) before cutting them with the cryostat. Make sure you are turning the instrument on ahead of time and reaching the temp you need to section them. Put the knife inside when you are turning on the machine so it has the same temp as that of the tissue block.
I usually lift the sections and put them in 12 or 24 tissue culture wells and store at 4 C.
Hope it helps. Please reach out if you have any further question on this.
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